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Factors affecting enzyme activity

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Effect of enzyme, substrate, product concentration, time, temperature, pH and presence of activators and inhibitors.

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Factors affecting enzyme activity

  1. 1. Factors affecting enzyme activity Namrata Chhabra 30-Mar-18 Namrata Chhabra 1
  2. 2. What are Enzymes ? • Enzymes are biological catalysts of the chemical reactions. • They are neither consumed nor permanently altered as a consequence of their participation in a reaction. • With the exception of catalytic RNA molecules, or ribozymes, all enzymes are proteins in nature. 30-Mar-18 Namrata Chhabra 2
  3. 3. Enzyme catalyzed reaction • The basic enzymatic reaction can be represented as follows: • where E represents the enzyme catalyzing the reaction, S the substrate, the substance being changed, and P the product of the reaction. 30-Mar-18 Namrata Chhabra 3
  4. 4. Enzyme Catalysis- overview 30-Mar-18 Namrata Chhabra 4
  5. 5. Thermodynamic perspective of mechanism of enzyme action • All enzymes accelerate reaction rates by providing transition states with a lowered activation energy for formation of the transition states. • The lower activation energy means that more molecules have the required energy to reach the transition state. 30-Mar-18 Namrata Chhabra 5
  6. 6. Thermodynamic changes-overview 30-Mar-18 Namrata Chhabra 6
  7. 7. Enzyme kinetics • The rate of a chemical reaction is described by the number of molecules of reactant(s) that are converted into product(s) in a specified time period. • International unit – One IU is defined as the activity of the enzyme which transforms one micro mole of substrate in to products per minute per litre of sample under optimal conditions and at defined temperature . It is expressed as IU/L • Katal – catalytic unit – One Katal is defined as the number of mole of substrate transformed per second per litre of sample. It is abbreviated as kat or k. 30-Mar-18 Namrata Chhabra 7
  8. 8. Numerous factors affect the reaction rate Reaction rate Substrate concentration Enzyme concentration Product concentration TemperaturepH Time Activators and inhibitors 30-Mar-18 Namrata Chhabra 8
  9. 9. Effect of substrate concentration • At lower concentrations, the active sites on most of the enzyme molecules are not filled because there is not much substrate. • Higher concentrations cause more collisions between the molecules. • The rate of reaction increases(First order reaction). 30-Mar-18 Namrata Chhabra 9
  10. 10. Effect of substrate concentration (contd.) • The maximum velocity of a reaction is reached when the active sites are almost continuously filled. • Increased substrate concentration after this point will not increase the rate. • Reaction rate therefore increases as substrate concentration is increased but it levels off (Zero order reaction) 30-Mar-18 Namrata Chhabra 10
  11. 11. Effect of substrate concentration The shape of the curve that relates activity to substrate concentration is hyperbolic. First order reaction Zero order reaction 30-Mar-18 Namrata Chhabra 11
  12. 12. • At points A or B, increasing or decreasing [S] therefore will increase or decrease the number of ES complexes with a corresponding change in vi. • The rate of reaction is substrate dependent (First order reaction). • At point C essentially all the enzyme is present as the ES complex. Since no free enzyme remains available for forming ES, further increases in [S] cannot increase the rate of the reaction . • Reaction rate therefore becomes independent of substrate concentration (Zero order reaction). 30-Mar-18 Namrata Chhabra 12
  13. 13. km and its significance • Michaelis constant, km is the substrate concentration at which V1 is half the maximal velocity (Vmax /2) attainable at a particular concentration of enzyme. • km thus has the dimensions of substrate concentration. • It is specific and constant for a given enzyme under defined conditions of time , temperature and p H • Km determines the affinity of an enzyme for its substrate, lesser the Km for is the affinity and vice versa • Km value helps in determining the true substrate for the enzyme. 30-Mar-18 Namrata Chhabra 13
  14. 14. Effect of enzyme concentration As the amount of enzyme is increases the rate of reaction increases. If there are more enzyme molecules than are needed, adding additional enzyme will not increase the rate. Reaction rate therefore increases as enzyme concentration increases but then it levels off. 30-Mar-18 Namrata Chhabra 14
  15. 15. Effect of product concentration • The enzyme activity declines when the products start accumulating. This is called product or feed back inhibition. • Under certain conditions reverse reaction may be favored forming back the substrate. 30-Mar-18 Namrata Chhabra 15
  16. 16. Effect of Temperature • Raising the temperature increases the kinetic energy of molecules. • Increasing the kinetic energy of molecules also increases their motion and therefore the frequency with which they collide. • This combination of more frequent and more highly energetic and productive collisions increases the reaction rate. 30-Mar-18 Namrata Chhabra 16
  17. 17. Temperature coefficient • The Q10, or temperature coefficient, is the factor by which the rate of a biologic process increases for a 10 °C increase in temperature. • For the temperatures over which enzymes are stable, the rates of most biologic processes typically double for a 10 °C rise in temperature (Q10 = 2). (A ten degree Centigrade rise in temperature will increase the activity of most enzymes by 50 to 100%). 30-Mar-18 Namrata Chhabra 17
  18. 18. Effect of Temperature • The reaction rate increases with temperature to a maximum level, then abruptly declines with further increase of temperature. • Most animal enzymes rapidly become denatured at temperatures above 40oC. 30-Mar-18 Namrata Chhabra 18
  19. 19. Effect of Temperature • The optimal temperatures of the enzymes in higher organisms rarely exceed 50 °C. • Some enzymes lose their activity when frozen. • Enzymes from thermophilic bacteria found in hot springs are active at 100 °C. • Changes in the rates of enzyme-catalyzed reactions that accompany a rise or fall in body temperature constitute a prominent survival feature for "cold-blooded" life forms such as lizards or fish, whose body temperatures are dictated by the external environment. 30-Mar-18 Namrata Chhabra 19
  20. 20. Effect of Hydrogen Ion Concentration (pH) • The rate of almost all enzyme- catalyzed reactions exhibits a significant dependence on hydrogen ion concentration. • Most intracellular enzymes exhibit optimal activity at pH values between 5 and 9. • When the activity is plotted against pH, a bell-shaped curve is usually obtained 30-Mar-18 Namrata Chhabra 20
  21. 21. Effect of Hydrogen Ion Concentration (pH) The relationship of activity to hydrogen ion concentration reflects the balance between enzyme denaturation at high or low pH The binding and recognition of substrate molecules with dissociable groups of enzymes typically involves the formation of salt bridges. The most common charged groups are the negative carboxylate groups and the positively charged groups of protonated amines. Gain or loss of critical charged groups thus will adversely affect substrate binding and thus will retard or abolish catalysis. 30-Mar-18 Namrata Chhabra 21
  22. 22. Effect of Hydrogen Ion Concentration (pH) • The pH optimum—i. e., the pH value at which enzyme activity is at its maximum—is often close to the pH value of the cells (i. e., pH 7). • There are also exceptions to this. • The proteinase pepsin , which is active in the acidic gastric lumen, has a pH optimum of 2, while alkaline phosphatase is active at pH values higher than 9 30-Mar-18 Namrata Chhabra 22
  23. 23. Effect of activators and Coenzymes • The activity of certain enzymes is greatly dependent on metal ion activators and coenzymes. • Vitamins act as coenzymes in a variety of reactions. 30-Mar-18 Namrata Chhabra 23
  24. 24. Effect of modulators and Inhibitors • The enzyme activity is reduced in the presence of an inhibitor and • the enzyme activity may be increased in the presence of a positive modifier. 30-Mar-18 Namrata Chhabra 24
  25. 25. Effect of Time • The longer an enzyme is incubated with its substrate, the greater the amount of product that will be formed. • However, the rate of formation of product is not a simple linear function of the time of incubation. • All proteins suffer denaturation, and hence loss of catalytic activity, with time. 30-Mar-18 Namrata Chhabra 25
  26. 26. Effect of Time • Enzyme catalyzed reactions undergo completion in an optimum time. • Variations of time affect the rates of enzyme catalyzed reactions. 30-Mar-18 Namrata Chhabra 26
  27. 27. Michaelis-Menten Kinetics • The Michaelis-Menten equation is a quantitative description of the relationship among the rate of an enzyme- catalyzed reaction [v1], the concentration of substrate [S] and two constants, Vmax and km (which are set by the particular equation). The symbols used in the Michaelis- Menten equation refer to the reaction rate [v1], maximum reaction rate (V max), substrate concentration [S] and the Michaelis-Menten constant (km). 30-Mar-18 Namrata Chhabra 27
  28. 28. Michaelis-Menten Kinetics • The dependence of initial reaction velocity on [S] and Km may be illustrated by evaluating the Michaelis-Menten equation under three conditions. • (1) When [S] is much less than km, the term km + [S] is essentially equal to km. Replacing Km + [S] with Km reduces equation to 30-Mar-18 Namrata Chhabra 28
  29. 29. When [S] is much less than km • Since V max and km are both constants, their ratio is a constant (k). • In other words, when [S] is considerably below km, V max is proportionate to k[S]. • The initial reaction velocity therefore is directly proportionate to [S]. 30-Mar-18 Namrata Chhabra 29
  30. 30. When [S] is much greater than km • ,the term km + [S] is essentially equal to [S]. Replacing km + [S] with [S] reduces equation to : Thus, when [S] greatly exceeds km, the reaction velocity is maximal (V max) and unaffected by further increases in substrate concentration. 30-Mar-18 Namrata Chhabra 30
  31. 31. (3) When [S] = km • Equation states that when [S] equals km, the initial velocity is half-maximal. • Equation also reveals that km can be determined experimentally from—the substrate concentration at which the initial velocity is half-maximal. 30-Mar-18 Namrata Chhabra 31
  32. 32. Linear Form of the Michaelis-Menten Equation • The direct measurement of the numeric value of V max and therefore the calculation of km often requires impractically high concentrations of substrate to achieve saturating conditions. • A linear form of the Michaelis-Menten equation circumvents this difficulty and permits V max and km to be extrapolated from initial velocity data obtained at less than saturating concentrations of substrate. 30-Mar-18 Namrata Chhabra 32
  33. 33. Linear Form of the Michaelis-Menten Equation invert Factor and simplify 30-Mar-18 Namrata Chhabra 33
  34. 34. Double reciprocal or Lineweaver-Burk plot • Equation is the equation for a straight line, y = ax + b, where y = 1/vi and x = 1/[S]. • A plot of 1/vi as y as a function of 1/[S] as x therefore gives a straight line whose y intercept is 1/ V max and whose slope is km / V max. • Such a plot is called a double reciprocal or Lineweaver-Burk plot 30-Mar-18 Namrata Chhabra 34
  35. 35. Carry home message Enzyme is affected by: Substrate concentration Product concentration Enzyme concentration Temperature Ph Time and Presence of activators and inhibitors 30-Mar-18 Namrata Chhabra 35
  36. 36. Thank you 30-Mar-18 Namrata Chhabra 36